Amoeboid locomotion, a fundamental property of many eukaryotic cells, plays a key role in a number of physiological processes including inflammation, would healing, neuronal targeting, and metastatic invasion. The purpose of this proposal is to investigate the molecular mechanism of amoeboid movement using the simple, specialized sperm of the nematode, Ascaris suum, as an experimental system. These cells display the same morphology and motile behavior as conventional crawling cells, but lack the complex actin-based on major sperm protein (MSP) filaments that assemble along the leading edge of the pseudopod and bundle into meshworks in a pattern that is tightly coupled to cell translocation. This locomotory apparatus has been reconstituted in vitro and used to show that assembly and bundling of MSP filaments can move membranes in the same way as the MSP cytoskeleton pushes the plasma membrane forward in crawling sperm. Dissection of this apparatus reveled that, in addition to MSP, motility requires (I) initiation of polymerization by the plasma membrane and (ii) a cytosolic component with properties of the Rho subfamily of GTPases. This powerful in vitro motility system will be used to identify and characterize the components of the locomotory apparatus. This information will then be integrated into the broader perspective of defining how the cellular machinery produces movement. Approaches that have been used to solve the structure of MSP will be extended to establish the precise interaction interfaces between MSP molecules that are responsible for their intrinsic polymerization and filament bundling properties. The in vitro assay will be used in conjunction with mutant MSPs, designed using the structural data and produced by protein engineering, to define the contribution of each class of interaction to polymerization and bundling. Biochemical and molecular methods will be used to identify and characterized the membrane and cytosolic components required for motility and explore how they contribute to cytosketletal dynamics and locomotion. Information derived from studies of individual mortality proteins, together with procedures designed to identify any additional components, will be used to reconstitute the locomotory machinery from its purified constituents, and so define its minimum molecular composition. Confocal microscopy will be used to established the distribution and dynamics of these components in vivo and provide a framework for integrating the molecular data into a cellular context. The long-term objective of this proposal is to determine the molecular mechanism of nematode sperm locomotion so that comparison of MSP-and actin-based systems can used to understand the basic principles of amoeboid cell motility.